Ammonia production and excretion by the kidney play important roles in the maintenance of acid-base homeostasis. Renal ammonia production and excretion are the major means by which the kidney protects the body against increased acid loads. Reductions in renal ammonia excretion lead to the accumulation of acid in the body. In addition to changes in acid-base status, other factors such as potassium balance and concentration and hormones may alter ammonia production and transport under normal and abnormal acid-base conditions. Because each portion of the nephron contribute differently to ammonia production and transport, studies to examine the regulation of ammonia production and transport by the nephron must utilize techniques which take into account the heterogeneity of the nephron. To examine the regulation of ammonia production and transport by specific nephron segments, the proposed research utilizes the in vitro microperfusion technique coupled with a sensitive assay for ammonia. The major advantages of this approach are that specific segments are examined under perfused conditions, that production and transport may be assessed in the same preparation, and that luminal and bath conditions may be controlled. Studies by the principal investigator using this technique clearly demonstrate its usefulness in the study of ammonia transport and production in response to changes in pH and potassium concentration and to certain hormones. In addition, the principal investigator has developed new cell lines derived from specific segments of the nephron of a Brinster SV40 transgenic mouse. These cell lines will be used to study ammonia production and transport. The major advantages of using cell lines from specific segments of the nephron are that their origin is clearly defined, that large numbers of cells may be generated, that cells are readily separated for whole-cell electrophysiologic studies, and that medium conditions may be controlled to determine the effects of more prolonged.changes in the extracellular environment on ammonia production and transport. Initial studies on Sl, S2, S3 proximal tubule cells and on thick ascending limb cells have demonstrated that several characteristics displayed by corresponding freshly dissected nephron segments are expressed by these cells including ph-induced changes in ammonia production in Sl and S2 cells and high apical ammonium transport in thick ascending limb cells. The proposed approaches are unique and complementary and represent major advances in the study of ammonia production and transport by tubular segments and cells of specific portions of the nephron. The proposed research will provide previously unobtainable information about the regulation of ammonia production and transport which will extend our knowledge about renal acid-base physiology.